L/3-,3^  ^* 


^QHAM/VII  agricultural  experiment  station, 

J      J.    M.  WESTGATE,  Agronomist  in  Charge. 


Bulletin    No.  39. 


THE  BIOCHEMICAL  DECOMPOSITION  OF 

NITROGENOUS  SUBSTANCES 

IN  SOILS. 


LIB. 

NTS  OEPT. 


U.S.  DEPOSITORY 


W.  P.  KELLEY, 

Chemist. 


UNDER  THE  SUPERVISION  OF 

STATES    RELATIONS     SERVICE. 
U,  S.  DEPARTMENT  OF  AGRICULTURE. 


*>^  ^*rH^$tv\^|ff  x- , 

~'<?<&¥*JWrffST   PRINTING   OFFICE. 

Vdl5. 


Issued  August  3,  1915. 

HAWAII  AGRICULTURAL  EXPERIMENT  STATION, 

J.    M.  WESTGATE,  Agronomist  in  Charge. 


Bulletin   No.  39. 


THE  BIOCHEMICAL  DECOMPOSITION  OF 

NITROGENOUS  SUBSTANCES 

IN  SOILS. 


BY 


W.  P.  KELLEY, 

Chemist. 


UNDER  THE  SUPERVISION  OF 

STATES    RELATIONS    SERVICE, 
U.  S.  DEPARTMENT  OF  AGRICULTURE. 


WASHINGTON: 

GOVERNMENT   PRINTING   OrFIOE. 
1915. 


HAWAII  AGRICULTURAL  EXPERIMENT  STATION,  HONOLULU. 

[Under  the  supervision  of  A.  C.  True,  Director  of  the  States  Relations  Service,  United  States  Depart- 
ment of  Agriculture.  ] 

Walter  H.  Evans,  Chief  of  Division  of  Insular  Stations,  Office  of  Experiment  Stations. 

STATION   STAFF. 

J.  M.  Westgate,  Agronomist  in  Charge. 

J.  Edgar  Higgins,  Horticulturist. 

W.  P.  Kelley,1  Chemist. 

D.  T.  Fullaway,  Entomologist. 

W.  T.  McGeorge,  Chemist. 

Alice  R.  Thompson,  Assistant  Chemist. 

V.  S.  Holt,  Assistant  Horticulturist. 

C.  A.  Sahr,  Assistant  in  Agronomy. 

F.  G.  Krauss,  Superintendent  of  Extension  Work. 

F.  A.  Clowes,  Superintendent  Hawaii  Substations. 

1  Resigned  October  27, 1914. 


LETTER  OF  TRANSMITTAL. 


Hawaii  Agricultural  Experiment  Station, 

Honolulu,  Hawaii,  September  H,  1914- 
Sir  :  I  have  the  honor  to  submit  herewith  and  recommend  for  pub- 
lication as  Bulletin  No.  39  of  the  Hawaii  Experiment  Station  a  paper 
on  The  Biochemical  Decomposition  of  Nitrogenous  Substances  in 
Soils,  by  W.  P.  Kelley,  chemist.  More  exact  information  is  needed 
on  the  sources  from  which  plants  draw  nitrogen  for  their  growth. 
This  bulletin  contains  the  results  of  a  study  of  the  percentages  of 
ammonia  derived  from  the  bacterial  decomposition  of  various  organic 
nitrogenous  substances  in  soils.  In  six  out  of  the  eight  substances 
used  in  this  study  the  basic  diamino  acid  nitrogen  was  found  to  be 
more  readily  converted  into  ammonia  than  was  the  nitrogen  of 
other  groups. 

Respectfully, 

E.  V.  Wilcox, 
Special  Agent  in  Charge. 
Dr.  A.  C.  True, 

Director  States  Relations  Service, 

V.  S.  Department  of  Agriculture,   Washington,  D.  C. 

Publication  recommended. 
A.  C.  True,  Director. 

Publication  authorized. 
D.  F.  Houston, 

Secretary  of  Agriculture. 
(3) 


CONTENTS. 


Page. 

Introduction 5 

Ammonification  under  varying  conditions 6 

Series  I — Ammonification  in  silica  sand 6 

Series  II — Ammonification  in  soil 8 

Series  III — Ammonification  of  equal  amounts  of  nitrogen 9 

Series  IV — Ammonification  in  soil  under  anaerobic  conditions 10 

Series  V — Ammonification  with  equal  amounts  of  nitrogenous  and  non- 
nitrogenous  materials 11 

Series  VI — Ammonification  with  varying  amounts  of  casein 12 

Series  VII — Ammonification  of  casein  during  different  lengths  of  time 13 

Series  VIII — Ammonification  of  casein  in  silica  sand 14 

Series  IX — Ammonification  and  hydrolysis  of  casein 15 

Effects  of  bacterial  action  on  different  groups  of  nitrogen  compounds 17 

Casein 18 

Dried  blood 18 

Soy  bean  cake  meal 19 

Cottonseed  meal 20 

Linseed  meal 20 

Coconut  meal 21 

Globulin  from  cottonseed  meal 21 

Zein  from  maize 22 

Summary 24 


ILLUSTRATION 


Page. 
Figure  1.  Diagram  showing  the  ammonification  of  different  amounts  of  casein.         13 

(4) 


THE  BIOCHEMICAL  DECOMPOSITION  OF  NITROG- 
ENOUS SUBSTANCES  IN  SOILS. 


INTRODUCTION. 

The  chemical  changes  produced  in  the  nitrogenous  substances  of 
soils  by  bacteria  are  of  great  importance.  As  is  well  known,  the  end 
products  are  ammonia,  nitrate,  and  free  nitrogen,  but  the  interme- 
diate steps  of  the  change,  which  are  probably  of  great  importance, 
are  only  imperfectly  understood.  It  appears  that  proteins  undergo 
progressive  decomposition  in  soils  similar  to  that  which  takes  place 
in  animal  digestion,  and  it  is  highly  probable  that  ammonification 
is  preceded  by  hydrolysis.  From  the  investigations  of  Schreiner  et 
al.1  it  has  been  shown  that  small  amounts  of  protein  and  nucleo- 
protein  cleavage  products  are  widely  distributed  in  soils ;  and  Lohnis 
and  Green  2  recently  found  from  ammonification  experiments  with 
flesh  meal  that  in  the  early  stages  of  the  action  greater  amounts  of 
ammonia  were  obtained  by  distilling  a  1  per  cent  hydrochloric  acid 
extract  with  caustic  soda  than  with  magnesia.  They  held  that  the 
caustic  soda  decomposed  soluble  hydrolytic  products  (amino  acids) 
split  off  by  the  bacteria.  At  a  later  stage,  when  the  amino  acids  had 
presumably  been  more  completely  decomposed,  the  yields  of  ammonia 
by  the  two  methods  were  more  nearly  equal. 

It  is  probable  that  not  all  of  the  nitrogen  in  a  given  protein  is 
equally  susceptible  to  ammonification  s  and  that  different  proteins 
undergo  decomposition  at  different  rates,  even  when  all  other  con- 
ditions are  equal.  In  ammonification  experiments,  for  example,  it 
is  seldom  that  more  than  from  50  to  60  per  cent  of  the  nitrogen 
added  is  recovered  as  ammonia,  and  different  nitrogenous  substances 
yield  ammonia  at  greatly  different  rates.  As  is  well  known,  the  main 
portion  of  the  nitrogen  in  soils  occurs  in  organic  forms  and  has  pre- 
viously existed  as  vegetable  protein.  After  the  organic  matter  of 
soils  has  been  acted  upon  by  bacteria  for  a  time  a  residue,  rich  in 
nitrogen,  and  commonly  called  humus,  is  left,  which  undergoes  further 
decomposition  at  a  very  slow  rate.  Moreover,  the  rate  of  formation 
of  ammonia  from  humus  appears  to  depend  to  some  extent  on  the 
conditions  under  which  the  humus  was  itself  formed.     Generally  acid 

"  U.  S.  Dept.  Agr.,  Bur.  Soils  Bdls.  53,  74,  80,  83,  88. 
iCentbl.  Bakt.  [etc.],  2.  AM.,  37  (1913),  pp.  534-562. 
»See  Jodidi,  Iowa  Sta.  Research  Bui.  9  (1912). 

(5) 


humus,  or  humus  formed  under  anaerobic  conditions,  is  thought  to 
decompose  more  slowly  than  neutral  humus.  Thus  it  seems  that 
conditions  arise  during  the  course  of  bacterial  action  which  tend  to 
check  the  process,  or  else  some  of  the  constituents  of  proteins  are  more 
difficult  of  hydrolysis  and  decomposition  than  others. 

Previous  investigations  on  proteins  throw  much  light  on  this  sub- 
ject. It  is  known  that  proteins  are  composed  of  a  number  of  amino 
acids  and  that  different  proteins  undergo  hydrolysis  by  acids,  alkalis, 
and  enzyms  at  different  rates,  yielding  varying  amounts  of  the  several 
amino  constituents.  From  limited  study  on  the  subject  it  seems 
that  bacterial  and  enzymatic  action  bring  about  similar  hydrolysis 
of  proteins,  with  the  important  difference  that  the  products  of 
hydrolysis  are  subject  to  decomposition  by  bacteria.  During  the 
course  of  such  action  certain  amino  acids  are  split  off  more  rapidly 
than  others.  Abderhalden  and  Reinbold  1  found,  for  example,  that 
in  the  tryptic  digestion  of  edestin  from  cotton  seed,  97.6  per  cent  of 
the  tyrosin  had  been  split  off  at  the  end  of  2  days,  while  only  7.4  per 
cent  of  the  glutaminic  acid  had  been  hydrolyzed. 

From  previous  work  in  this  laboratory  2  it  has  been  shown  that 
Hawaiian  soils  contain  relatively  greater  amounts  of  amid  and 
smaller  amounts  of  basic  (diamino  acid)  nitrogen  than  the  vegetable 
proteins.  From  this  it  has  been  suggested  that  a  study  of  the  group 
changes  produced  under  the  action  of  bacteria  might  throw  important 
light  on  both  the  availability  of,  and  the  nature  of  bacterial  action 
on,  nitrogenous  fertilizers.  Various  factors,  such  as  the  degree  of 
aeration,  the  acidity  of  the  medium,  the  carbohydrates  present,  the 
synthesis  of  proteins  in  the  body  cells  of  the  bacteria,  the  absorption 
of  organic  nitrogen  compounds  in  varying  degrees  by  plants,  etc.,  so 
complicate  the  problem  as  to  render  very  difficult  an  interpretation 
of  the  chemistry  of  bacterial  action  in  soils.  It  has  been  suggested, 
however,  that  by  also  studying  the  rates  of  decomposition  under 
varying  conditions  some  light  might  be  thrown  on  this  question. 

AMMONIFICATION  UNDER  VARYING  CONDITIONS. 

SERIES    I — AMMONIFICATION   IN    SILICA    SAND. 

The  materials  used  in  this  investigation  contained  the  following 
percentages  of  nitrogen:  Casein  (Eimer  and  Amend),  12.40  per  cent; 
dried  blood,  13.29  per  cent;  soy  bean  cake  meal,  8.28  per  cent;  cot- 
tonseed meal,  5.10  per  cent;  linseed  meal,  5  per  cent. 

In  order  to  insure  maximum  aeration  the  first  series  of  tests  was 
conducted  with  the  use  of  silica  sand.  One  gram  each  of  the  above 
nitrogenous  materials  and  one  gram  of  calcium  carbonate  were  thor- 
oughly mixed  with  100-gram  portions  of  sand  in  tumblers.     Ten  cubic 

i  Ztschr.  Physiol.  Chem.,  46  (1905),  pp.  159-175.  *  Hawaii  Sta.  Bui.  33  (1914). 


centimeters  of  an  infusion,  made  by  shaking  for  ten  minutes  200  grams 
of  fresh  soil,  taken  from  the  citrus  orchard  of  this  station,  with  250 
cubic  centimeters  of  sterile  water  was  then  added  to  each  beaker  and 
thoroughly  mixed  with  the  contents.  The  beakers  were  covered  with 
watch  glasses  and  the  mixtures  incubated  at  28°  C.  The  ammonia 
was  determined  at  intervals,  as  shown  in  the  table,  by  three  different 
methods.  One  portion  was  distilled  directly  with  magnesia;  three 
other  portions  were  first  shaken  with  250  cubic  centimeters  of  1  per 
cent  hydrochloric  acid,  allowed  to  stand  for  an  hour,  and  then  filtered. 
An  aliquot  of  one  of  these,  corresponding  to  75  per  cent  of  the  mate- 
rial used,  was  made  alkaline  with  magnesia  and  distilled,  while 
aliquots  of  the  other  two  portions  were  distilled  with  caustic  soda. 
The  results  obtained  were  as  follows: 

Amount  of  ammonia  formed  in  silica  sand  from  different  substances. 


Method  of  determination. 

Nitrogen  as  ammonia  from— 

Period  of  incu- 
bation. 

Casein. 

Dried 
blood. 

Soy  bean 
cake 
meal. 

Cotton- 
seed 
meal. 

Linseed 
meal. 

[Distilled  directly  with  MgO 

iHCl  solution  distilled  with  MgO  . . 

JHC1  solution  distilled  with  NaOH  . 

(Distilled  directly  with  MgO 

1 HC1  solution  distilled  with  MgO  . . 

[HCl  solution  distilled  with  NaOH  . 

[Distilled  directly  with  MgO 

1  HCl  solution  distilled  with  MgO  . . 

[HCl  solution  distilled  with  NaOH . 

/HCl  solution  distilled  with  MgO  . . 
\HC1  solution  distilled  with  NaOH  . 

Mg. 

49.0 

47.2 

/        44.4 

\        42.0 

52.1 

44.8 

/        44.8 

\        41.2 

Lost 

41.1 

/        35.5 

\        34.4 

25.2 

24.8 

Mg. 

4.5 

2.8 

4.2 

4.2 

14.3 

15.0 

15.9 

15.0 

26.0 

24.9 

Lost 

29.1 

19.8 

23.5 

Mg. 
13.4 
12.0 
9.9 
10.3 
27.9 
20.5 
22.4 
22.6 
21.7 
20.4 
20.2 
20.2 
13.0 
19.6 

Mg. 
6.2 
4.8 
4.3 
4.1 
12.7 
12.3 
12.5 
11.6 
12.9 
10.4 
10.4 
12.3 
11.2 
9.9 

Mg. 
7.3 
5.6 

6.4 
5.6 
16.2 
13.1 

6  days 

14.9 
14.7 
17.9 
14.2 

9  days 

15.9 
16.8 
14.9 

14.7 

The  different  materials  were  converted  into  ammonia  at  greatly 
different  rates.  Casein  was  the  most  readily  ammonified,  the  con- 
centration of  ammonia  having  reached  a  maximum  at  the  end  of 
two  days,  when  about  40  per  cent  of  the  nitrogen  had  been  converted 
into  ammonia.  After  four  days  the  ammonia  content  became  con- 
siderably reduced.  In  view  of  the  yields  of  ammonia  found  in  experi- 
ments with  soil  (Series  II)  it  is  probable  that  ammonification  was 
active  for  longer  than  two  days,  but  then  evaporation  and  nitrifica- 
tion more  than  equaled  ammonification.  The  rapidity  with  which 
casein 1  was  ammonified  as  compared  with  the  other  materials  is 
especially  interesting. 

The  formation  of  ammonia  from  dried  blood  took  place  slowly 
during  the  first  two  days,  but  later  the  yield  approached  that  from 
casein.     Soy  bean   cake   meal  was   ammonified  considerably  more 

i  See  Brown,  Iowa  Sta.  Research  Bui.  11;  Centbl.  Bak„.  [etc.],  2.  Abt.,  39  (1913),  pp.  61-73. 
96280°— 15 2 


8 

rapidly  during  the  first  four  days  than  dried  blood,  after  which  time 
the  yields  decreased;  while  cottonseed  meal  and  linseed  meal  each 
gave  lower  yields,  the  highest  concentration  of  ammonia  from  the 
former  being  on  the  fourth  day  and  from  the  latter  on  the  sixth  day. 
Direct  distillation  with  magnesia  after  two  days  yielded  slightly 
more  ammonia  in  most  instances  than  the  other  methods.  Later 
the  results  by  the  different  methods  were  fairly  concordant.  With 
only  a  few  exceptions,  as  large  amounts  of  ammonia  were  obtained 
by  distilling  the  hydrochloric  acid  extracts  with  magnesia  as  with 
caustic  soda.a  The  above  data  therefore  give  but  little  indication 
that  hydrolysis  was  more  rapid  than  ammonification. 


SERIES    II — AMMONIFICATION    IN    SOIL. 

This  series  of  experiments  was  conducted  with  the  fresh  heavy 
clay  soil  from  which  the  infusions  were  made  in  the  previous 
series.  One-hundred-gram  portions  were  each  thoroughly  mixed 
with  1  gram  of  the  nitrogenous  materials  and  1  gram  of  calcium  car- 
bonate and  placed  in  tumblers.  Sterile  water  was  added  so  as  to 
bring  the  moisture  up  to  25  per  cent.  After  incubating  at  28°  C. 
the  ammonia  was  determined  as  in  the  previous  series.  On  the  ninth 
day  the  nitrate  was  also  determined  in  separate  portions  by  the  phenol 
disulphonic  acid  method,  and  was  added  to  the  average  ammonia  at 
this  period  in  estimating  the  total  ammonification. 

Amount  of  ammonia  formed  in  soil  from  different  substances. 


Method  of  determination. 

Nitrogen  as  ammonia  from— 

Period  of  incu- 
bation. 

Casein. 

Dried 
blood. 

Soy  bean 
cake 
meal. 

Cotton- 
seed 
meal. 

Linseed 
meal. 

2  days 

Distilled  directly  with  MgO 

HCl  solutions  distilled  with  MgO.. 

HCl  solutions  distilled  with  NaOH 

Distilled  directly  with  MgO 

HCl  solutions  distilled  with  MgO.. 

HCl  solutions  distilled  with  NaOH 

Distilled  directly  with  MgO 

HCl  solutions  distilled  with  MgO.. 

HCl  solutions  distilled  with  NaOH 

/HCl  solutions  distilled  with  MgO.. 
\HC1  solutions  distilled  with  NaOH 

Nitrate  N 

Mg. 

50.8 

38.1 

/        40.0 

\        40.3 

57!  7 

/        65.5 

\        60.5 

68.6 

59.1 

/        56.6 

\        56.8 

59.1 

56.8 

Mg. 

4.1 

2.2 

4.2 

4.5 

24.1 

18.5 

21.3 

21.6 

42.4 

40.9 

33.6 

35.2 

<>38.9 

49.0 

Mg. 

7.8 

5.6 

8.1 

7.8 

21.8 

20.4 

17.4 

19.9 

29.4 

26.3 

28.3 

23.8 

26.6 

27.2 

Mg. 
5.6 
4.8 
4.2 
5.6 

11.5 
9.5 
8.1 

10.1 

10.6 
9.2 

10.6 
9.8 
7.8 
7.3 

Mg. 
2.0 

4  days 

4.2 
2.8 
11.2 
9.5 

6  days 

9.5 
10.6 
13.9 
10.9 

9  days 

10.9 
11.2 
7.0 

9.8 

4.4 
62.3 
50.2 

7.4 

56.4 
42.4 

7.0 
33.9 
40.9 

6.3 
13.8 
27.1 

4.6 

9  days 

Total  ammonification 

Per  cent  of  total  N  converted 
into  NH3. 

13.0 

26.0 

o  It  is  not  surprising  that  practically  as  much  ammonia  was  obtained  by  distilling  the  solutions  with 
magnesia  as  with  caustic  soda,  since  the  acid  amids  are  decomposed  by  each  alike,  and  only  two  other 
protein  cleavage  products,  cystin  and  arginin,  yield  ammonia  to  caustic  soda.  Cystin  occurs  in  very  small 
amounts  in  most  proteins. 

b  Not  used  in  average. 


The  amounts  of  ammonia  recovered  from  the  different  materials 
after  two  days  and  the  relative  rates  of  ammonincation  throughout 
bear  similar  relations  to  those  of  the  previous  series.  In  the  cases 
of  casein,  dried  blood,  and  soy  bean  cake  meal,  from  which  the  largest 
yields  of  ammonia  were  obtained  in  the  sand  cultures,  still  greater 
amounts  accumulated  in  the  soil.  The  ammonincation  of  cotton- 
seed meal  and  linseed  meal  was  more  nearly  equal  at  each  period, 
being  practically  the  same  as  that  which  took  place  in  sand.  The 
losses  by  evaporation  were  considerably  less  than  from  the  sand,  due 
probably  to  the  high  absorbing  power  of  the  clay.  A  small  amount 
of  nitrification  took  place,  but  it  was  not  proportional  to  ammonifica- 
tion.  The  data  for  the  total  ammonincation  show  a  wide  difference 
in  the  decomposition  of  the  materials;  50.2  per  cent  of  the  nitrogen 
in  casein  was  converted  into  ammonia,  42.4  per  cent  in  dried  blood, 
40.9  per  cent  in  soy  bean  cake  meal,  27.1  per  cent  in  cottonseed 
meal,  and  26  per  cent  in  linseed  meal.  Thus  the  availability  of  these 
materials  as  measured  by  ammonincation  varies  greatly. 

Direct  distillation  with  magnesia,  especially  in  those  instances 
where  comparatively  large  amounts  of  ammonia  had  accumulated, 
yielded  considerably  more  ammonia  than  the  distillation  of  hydro- 
chloric-acid extracts;  but,  again,  distillation  of  the  latter  with  mag- 
nesia yielded  as  much  ammonia  as  distillation  with  caustic  soda." 
It  would  seem,  therefore,  that  direct  distillation  with  magnesia 
affords  a  truer  measure  of  ammonincation  in  clay  soils  than  indirect 
distillation  of  hydrochloric- acid  solutions.  In  all  of  the  subsequent 
series  reported  in  this  bulletin  the  former  method  was  used. 

SERIES   III — AMMONIFICATION  OF  EQUAL  AMOUNTS   OF  NITROGEN. 

In  this  series  the  nitrogenous  materials  were  added  so  as  to  furnish 
equal  amounts  of  nitrogen  (132.9  milligrams).  These  and  1  gram  of 
calcium  carbonate  were  mixed  with  100  grams  of  the  same  fresh  soil. 
After  bringing  the  moisture  content  to  25  per  cent  and  incubating 
as  before  the  yields  of  ammonia  were  as  follows: 

Amount  of  ammonia  from  equal  amounts  of  nitrogen. 
[Average  of  2  samples.] 


Period  of  incubation. 


2  days 

4  days 

6  days 

9  days 

Per  cent  of  total  N  converted  into  NH3 


Nitrogen  as  ammonia  from- 


Casein, 
L.072gm. 


Mg. 
58.3 
74.9 
75.7 
75.2 


56.9 


Dried 

blood, 

1.000  gm. 


Mg. 

4.0 

38.5 

59.6 

65.6 


49.3 


Soybean 
cake 
meal, 

1.605  gm. 


Mg. 
14.9 
47.1 
64.8 
58.6 


48.7 


Cotton- 
seed 
meal, 
2.606  gm. 


Mg. 
17.3 
37.9 
42.6 
40.3 


32. 0 


Linseed 

meal, 

2.658  gm. 


Mg. 

3.5 

32.9 

39.9 

46.0 

34.6 


10 

The  foregoing  data  show  that  when  equal  amounts  ox  nitrogen 
were  added,  the  amounts  of  ammonia  formed  still  varied  considerably. 
Casein  was  far  more  readily  decomposed  during  the  early  stages  of 
the  action  than  the  other  materials.  At  the  end  of  2  days  58.3  milli- 
grams of  casein  nitrogen  had  been  ammonified,  as  contrasted  with  4 
milligrams  in  dried  blood,  14.9  milligrams  in  soy  bean  cake  meal, 
17.3  milligrams  in  cottonseed  meal,  and  3.5  milligrams  in  linseed 
meal.  Later  the  yields  became  more  nearly  equal  and  showed  much 
less  variation  than  when  equal  weights  of  the  materials  were  added 
(see  Series  II).  The  maximum  percentages  of  ammonia  formed 
from  the  different  materials,  not  allowing  for  evaporation  and  nitri- 
fication, the  latter  of  which  was  small,  ranged  from  56.9  per  cent 
from  casein  to  32  per  cent  from  cottonseed  meal. 


SERIES  IV — AMMONIFICATION  IN  SOIL  UNDER  ANAEROBIC  CONDITIONS. 

In  order  to  measure  the  rates  of  ammonification  under  anaerobic 
conditions  the  same  soil  was  used  and  sufficient  sterile  water  added 
to  insure  complete  submergence.  Equal  amounts  of  nitrogen  (132.9 
milligrams)  and  1  gram  of  calcium  carbonate  were  mixed  with  100- 
gram  portions  of  soil  as  in  the  preceding  series. 

Amount  of  ammonia  in  soil  under  anaerobic  conditions. 
[Average  of  2  samples.] 


Nitrogen  as  ammonia  from — 

Period  of  incubation. 

Casein 
1.072  gm. 

Dried 
blood 
lgm. 

Soy  bean 
cake  meal 
1.606  gm. 

Cotton- 
seed meal 
2.605  gm. 

Linseed 

meal 
2.658  gm. 

Mg. 

8.5 

47.3 

62.4 

70.7 

Mg. 
2.0 
6.6 
13.7 
16.3 

Mg. 
3.5 
9.1 
14.2 
18.6 

Mg. 
6.3 
8.2 
9.4 
11.4 

Mg. 
2.0 

3.5 

7.3 

9  days 

9.2 

Per  cent  of  total  N  converted  into  NH3 

53.2 

12.3 

14.0 

8.5 

6.9 

The  above  data  show  that  under  anaerobic  conditions  active 
ammonification  of  casein  did  not  begin  until  after  two  days'  stand- 
ing, but  it  then  was  approximately  as  rapid  as  under  aerobic  condi- 
tions. With  dried  blood,  soy  bean  cake  meal,  cottonseed  meal,  and 
linseed  meal  ammonification  took  place  at  greatly  reduced  rates 
throughout  the  experimental  period.  The  percentages  of  the  total 
nitrogen  converted  into  ammonia  were  as  follows:  Casein  53.2  per 
cent,  dried  blood  12.3  per  cent,  soy  bean  cake  meal  14  per  cent, 
cottonseed  meal  8.5  per  cent,  and  linseed  meal  6.9  per  cent.  By 
comparing  these  data  with  the  preceding  it  will  be  seen  that  anaerobic 
conditions  greatly  retarded  the  formation  of  ammonia  from  all  the 


11 

materials  except  casein.  As  is  well  known,  a  wide  range  of  organ- 
isms, including  bacteria  and  fungi,  have  the  power  of  splitting 
ammonia  from  organic  materials.  Some  of  these  are  aerobic,  some 
anaerobic,  and  others  facultative  anaerobic.  When  anaerobic  con- 
ditions are  brought  about,  the  formation  of  ammonia  has  usually 
been  found  to  be  considerably  less  than  under  aerobic  conditions. 

SERIES   V — AMMONIFICATION  WITH    EQUAL  AMOUNTS  OF  NITROGENOUS 
AND    NONNITROGENOUS    MATERIALS. 


In  order  to  make  the  conditions  for  bacterial  action  more  nearly 
equal,  the  different  materials  were  added  so  as  to  furnish  equal 
amounts  of  nitrogen  (132.9  milligrams),  and  the  inequalities  in  the 
amounts  of  nonnitrogenous  materials  were  balanced  by  the  addi- 
tion of  the  proper  amounts  of  cornstarch.  One  gram  of  calcium 
carbonate  and  100-gram  portions  of  the  above  soil  were  used.  Opti- 
mum moisture  conditions  were  provided  and  the  same  methods 
employed  as  in  the  previous  series. 

Amount  of  ammonia  formed,  with  equal  amounts  of  nitrogenous  and  nonnitrogenous 

materials. 

[Average  of  2  samples.] 


Period  of  incubation. 


Nitrogen  as  ammonia  from — 


Casein  1.072 

gm.,  starch 

1.586  gm. 


Dried  blood 

lgm.,  starch 

1.058  gm. 


Soy  bean 
cake  meal 
1.605  gm., 

starch 
1.053  gm. 


Cottonseed 
meal  2.606 
gm. ,  starch 
0.052  gm. 


Linseed 

meal 
2.658  gm. 


2  days 

4  days 

6  days 

9  days 

Per  cent  of  total  N  converted  into 
NH, 


Mg. 


31.0 

30.9 
35.8 
41.8 


Mg. 


0.3 

3.3 

10.4 

25.2 


Mg. 

4.4 

20.1 

33.5 

45.4 


Mg. 
10.3 
34.8 
42.6 
45.2 


Mg. 


2.4 
25.8 
36.2 
45.3 


31.4 


18.9 


34.1 


34.0 


34.1 


By  comparing  the  above  with  Series  III  it  will  be  seen  that  the  addi- 
tion of  starch  materially  influenced  the  accumulation  of  ammonia. 
By  adding  1.586  grams  of  starch  the  ammonification  of  casein  was 
reduced  practically  50  per  cent  throughout  the  nine  days.  The  effects 
on  the  ammonification  of  dried  blood  were  still  more  marked.  At 
the  end  of  two  days  practically  no  ammonia  had  appeared  and  the 
depressing  effect  was  observed  throughout  the  experimental  period; 
after  nine  days  only  18.9  per  cent  of  the  nitrogen  added  as  dried 
blood  occurred  in  the  form  of  ammonia.  A  similar,  although  less 
marked  depressing  effect,  was  observed  with  soy  bean  cake  meal, 
but  it  must  be  remembered  that  0.605  gram  less  starch  was  added 
than  with  the  dried  blood.     The  final  yields  of  ammonia  from  soy 


12 

bean  cake  meal,  cottonseed  meal,  and  linseed  meal  were  almost  the 
same,  being  equal  to  34.1  per  cent  of  the  total  nitrogen  added.  Thus 
it  is  shown,  in  common  with  the  findings  of  others,  that  the  carbon 
nitrogen  ratio  may  greatly  affect  the  accumulation  of  ammonia  in 
soils. 

The  low  yields  of  ammonia  in  the  presence  of  carbohydrates  have 
been  attributed  to  stimulation  of  the  ammonia-consuming  organ- 
isms, whereby  ammonia  is  reconverted  into  organic  forms,  and  to  the 
formation  of  metabolic  by-products,  probably  of  an  acid  nature, 
which  exercise  an  inhibitory  influence  on  ammoniflcation;  but,  as 
shown  by  Lipman  et  al.,1  the  depressing  effect  of  carbohydrates 
can  not  be  prevented  by  adding  calcium  carbonate.  It  is  probable 
that  the  energy  derived  by  the  ammonifying  organisms  themselves 
from  the  nonnitrogenous  matter  is  of  considerable  importance.  It 
is  true  these  organisms  can  satisfy  their  energy  requirements  from 
amino  compounds  of  various  sorts,  but  it  does  not  follow  that  a  part 
of  it  could  not  be  derived  more  advantageously  from  carbohydrates.2 
The  fact  that  bacteria  split  off  ammonia  from  nitrogenous  substances, 
thus  eliminating  a  portion  of  the  nitrogen  present,  is  in  itself  evidence 
of  their  demand  for  nonnitrogenous  matter. 

It  should  be  borne  in  mind  that  the  nonnitrogenous  constituents 
of  the  above  materials  were  made  up  of  different  chemical  compounds 
including  fats  and  carbohydrates.  The  exact  effect  of  fats  is  not 
known,  but  different  carbohydrates  produce  widely  different  effects. 
In  general,  soluble  carbohydrates  more  markedly  depress  ammonifl- 
cation than  insoluble  forms.3  The  nature  of  nitrogenous  constitu- 
ents in  these  materials  also  differs  considerably,  and  the  rate  at 
which  they  undergo  hydrolysis,  with  the  exception  of  casein,  has  not 
been  extensively  studied.  As  will  be  shown  later,  the  products  of 
acid  hydrolysis  vary  considerably.  Since  hydrolysis  is  probably 
essential  as  preliminary  to  ammoniflcation  4  any  differences  in  the 
rates  of  hydrolysis  would  probably  be  reflected  in  the  rates  of  ammoni- 
flcation. 

SERIES    VI AMMONIFICATION    WITH    VARYING    AMOUNTS     OF     CASEIN. 

In  the  preceding  series  the  yields  of  ammonia  from  practically  the 
same  amounts  of  casein  varied  from  50.2  per  cent  to  56.9  per  cent  of 
the  total  nitrogen  added.     In  the  following  series  the  concentration 
-of  casein  was  varied  and  the  same  amounts  of  soil  and  calcium  car- 
bonate were  used  throughout.     For  the  purpose  of  reducing  evapo- 

i  New  Jersey  Stas.  Rpt.  1911,  pp.  193-212. 

2  See  J.  G.  Lipman  et  al.,  New  Jersey  Stas.  Rpt.  1909,  pp.  166-169. 
a  See  Lipman  et  al.,  New  Jersey  Stas.  Rpt.  1911,  pp.  193-212. 

<  The  fact  that  peptone  has  frequently  been  found  to  ammonify  more  rapidly  than  dried  blood  or  cotton- 
seed meal  may  be  due  in  part  to  its  being  a  partially  hydrolyzed  substance. 


13 

ration  losses,  1  gram  of  monomagnesium  phosphate  was  al-^o  added, 
as  recommended  by  Lohnis  and  Green.1  After  bringing  the  moisture 
to  about  25  per  cent  and  incubating  at  28°  C.  for  four  days,  the 
ammonia  was  determined  as  before  with  the  following  results: 

Amount  of  ammonia  formed  from  varying  quantities  of  casein. 
[Average  of  2  samples.] 


Amount  of 
casein 
added. 

N  found  as 

Per  cent  of 
total  N  re- 

Amount of 
casein 
added. 

N  found  as 

Per  cent  of 
total  N  re- 

ammonia. 

covered  as 
NH3. 

ammonia. 

covered  as 
NH3. 

Gm. 

Mg. 

Gm. 

Mg. 

0.2 

12.0 

48.4 

1.5 

'111.7 

60.1 

.4 

25.5 

51.4 

2.0 

151.0 

60.9 

.6 

43.5 

58.2 

2.5 

192.3 

62.0 

.8 

58.2 

58.7 

3.0 

244.1 

65.9 

1.0 

70.7 

57.0 

1  1  sample  only. 

The  percentages  of  total  nitrogen  recovered  as  ammonia  increased 
as  the  amount  of  casein  increased,  varying  from  48.4  per  cent  with 

0.2  gram  to  65.9  per 
cent  with  3  grams  (see 
fig.  1).  Loss  of  am- 
monia by  evaporation 
was  not  important, 
since  the  percentage 
yields  were  greatest 
where  the  greatest 
concentration  of  am- 
monia occurred. 
Since  almost  no  nitri- 
fication took  place  in 
any  instance,  it  seems 
reasonable  to  believe 
that,  as  the  amount 
of  casein  added  is  in- 
creased, a  decreasing  percentage  of  the  total  nitrogen  present  would 
be  consumed  by  the  bacteria  and  consequently  higher  percentage 
yields  of  ammonia  be  obtained.  The  yield  from  1  gram  (57  per  cent) 
agrees  closely  with  that  recovered  in  preceding  series. 

SERIES  VII AMMONIFICATION  OF  CASEIN  DURING  DIFFERENT  LENGTHS 

OF   TIME. 

This  series  was  begun  at  the  same  time  as  Series  VI,  using  the  same 
soil.  One  gram  each  of  casein,  calcium  carbonate,  and  magnesium 
phosphate  was  mixed  with   100-gram  portions  of  soil,  sterile  water 


60 
50 
40 
30 
SO 
/O 

' 

7    O. 

2    0 

a  a 

6  a 

e  l 

o           /.. 

r          ?.< 

?              2. 

5           3.0 

Sj  G&AA4S  OF  CWS£W  /=VP£S£NT 

Fig.  1.— Diagram  showing  the  ammonification  of  different  amounts 
of  casein. 


i  Loc.  cit. 


14 

added,  and  the  mixture  incubated  as  before.  After  four  and  eight 
days,  respectively,  an  additional  gram  of  casein  was  added  in  certain 
instances  and  the  ammonia  and  nitrate  determined,  as  shown  in  the 
following  table: 

Amount  of  nitrogen  recovered  from  casein. 
[Average  of  2  samples.] 


Amount  of  casein  and  period  of  incubation. 


N  recovered  N  recovered 
asNH3.         asN03, 


Per  cent  of. 

total  N 
converted 
intoNHs. 


1  gram  4  days 

1  gram  8  days 

1  gram  12  days 

1  gram  4  days+1  gram  4  days  additional 

1  gram  4  days+1  gram  8  days  additional 

1  gram  4  days+1  gram  4  days  additional+1  gram  4  days 


Mg. 

72.1 

74.2 

60.2 

150.9 

138.9 

226.8 


Mg. 


12.5 

"e.'e' 

3.3 


57.3 
59.8 
58.6 
60.8 
57.1 
61.8 


Practically  the  same  amounts  of  ammonia  were  formed  from  a 
given  amount  of  casein  in  four  days  as  in  longer  periods.  The  yields 
from  1  gram  were  57.3  per  cent  in  four  days,  59.8  per  cent  in  eight 
days,  and  58.6  per  cent  in  twelve  days.  By  adding  another  gram 
on  the  fourth  day  and  allowing  the  action  to  continue  four  days 
longer  60.8  per  cent  of  the  total  nitrogen  was  converted  into  ammonia. 
The  portions  treated  in  the  same  way  but  allowed  to  stand  eight  days 
longer  gave  57.1  per  cent  yield  of  ammonia.  Finally  when  1  gram 
was  added  at  the  beginning  and  after  four  and  eight  days,  respectively, 
61.8  per  cent  of  the  total  nitrogen  was  converted  into  ammonia. 

The  above  data  show,  therefore,  in  common  with  the  preceding 
series,  that  increasing  percentages  of  the  total  nitrogen  were  converted 
into  ammonia  when  increasing  amounts  of  casein  up  to  3  grams  were 
acted  upon,  but  whether  this  fact  was  due  to  partial  suppression  of 
the  nonammonifying  organisms  can  not  be  positively  stated.  There 
is  evidence,  however,  that  under  the  conditions  of  these  experiments 
the  organisms  feed  on  the  organic  nitrogen  of  casein  rather  than  on  the 
ammonia  after  it  has  been  formed. 


SERIES    VIII— AMMONIFICATION    OF    CASEIN    IN    SILICA    SAND. 

The  amounts  of  ammonia  recovered  from  casein  in  the  preceding 
experiments  usually  did  not  exceed  60  per  cent  of  the  nitrogen  added, 
and  reached  a  maximum  point  by  the  fourth  day.  In  order  to  throw 
further  light  on  this  subject  a  series  of  experiments  was  carried  out 
with  silica  sand,  ^provision  being  made  for  absorbing  whatever 
ammonia  was  volatilized.  The  decomposition  took  place  in  closely 
stoppered  bottles  through  which  a  slow  current  of  air  was  drawn  by 
means  of  a  suction  pump.  The  current  of  air  was  first  drawn  through 
sulphuric  acid  to  remove  traces  of  ammonia  and  after  passing  through 


15 

the  bottles  was  again  drawn  through  a  solution  of  sulphuric  acid. 
Casein  prepared  by  Hammarsten  was  employed.  This  was  further 
purified  by  first  dissolving  in  tenth-normal  sodium  hydroxid  solution, 
then  precipitating  with  1  per  cent  acetic  acid,  filtering,  and  thoroughly 
washing'  with  alcohol  and  ether.  After  drying  in  vacuum  over 
sulphuric  acid,  the  product  was  found  to  contain  only  13.50  per  cent 
nitrogen,  showing  that  considerable  impurities  still  remained.  (Pure 
casein  contains  15.62  per  cent  N.) 

Five  grams  of  the  casein  was  mixed  with  500  grams  of  silica  sand 
and  optimum  moisture  brought  about  by  adding  50  cubic  centimeters 
of  a  soil  infusion  prepared  as  in  Series  I.  After  incubating  at  28°  C. 
for  one  week  the  contents  of  the  bottles  were  acidified  with  1  per  cent 
acetic  acid,  thoroughly  shaken,  filtered,  and  washed.  The  residue 
was  then  extracted  with  tenth-normal  sodium  hydroxid  and  the 
alkaline  solutions  acidified  with  1  per  cent  acetic  acid.  No  precipitate 
was  formed  and  the  solutions  were  combined  with  those  above.  Am- 
monia was  determined  in  the  solutions,  sand  residues,  and  the 
sulphuric  acid  by  distilling  with  an  excess  of  magnesia. 

Total  amount  of  ammonia  formed  from  casein  in  silica  sand. 


Determination  No. 

NasNH3 

soluble 

in  acetic 

acid. 

NasNH3 
in  the 
residue. 

NasNH3 
volatil- 
ized. 

Total 

yield 

01NH3. 

Per  cent  of 
total  N  con- 
verted into 
ammonia. 

1 

Mg. 

271.6 
273.0 
324.8 

Mg. 

96.6 
96.6 
55.  7 

Mg. 

62.6 
76.4 
43.4 

Mg. 
430.8 
446.0 
423.9 

63.8 

2 

66.1 

3 

62.8 

A  slightly  higher  yield  of  ammonia  was  obtained  than  previously, 
the  average  being  64.2  per  cent  of  the  total  nitrogen.  Since  no  casein 
could  be  precipitated  from  sodium  hydrate  solutions  after  bacterial 
action,  the  conclusion  that  the  entire  amount  of  casein  added  had 
undergone  hydrolysis  is  justified.  The  nature  of  the  undetermined 
balance  remains  to  be  determined. 


SERIES    IX AMMONIFICATION    AND    HYDROLYSIS    OF    CASEIN. 

The  purpose  of  this  series  was  to  study  the  relations  between  the 
rates  of  ammonification  and  hydrolysis  of  casein.  A  stock  solution 
of  casein  purified  as  in  the  previous  series  was  prepared  by  dissolving 
15  grams  in  150  cubic  centimeters  of  tenth-normal  sodium  hydroxid, 
then  diluting  to  2,100  cubic  centimeters.  One  hundred  cubic  centi- 
meter portions  were  placed  in  300  cubic  centimeter  Erlenmeyer  flasks 
and  10  cubic  centimeters  of  soil  infusion  added.  After  shaking,  the 
flasks  were  loosely  stoppered  with  cotton  plugs  and  incubated  at 
28°  C.     The  rate  of  hydrolysis    was  measured  by  precipitating  the 


16 

unhydrolyzed  casein  at  intervals  with  1  per  cent  acetic  acid,  then 
making  total  nitrogen  determinations  in  the  precipitates  as  recom- 
mended by  Walters.1  Ammonia  was  determined  in  the  filtrates  by 
distilling  with  an  excess  of  magnesia.  In  order  to  have  a  check  on 
autohydrolysis,  which  is  known  to  take  place  in  solutions  of  casein, 
a  number  of  the  flasks  were  set  aside  without  the  addition  of  infu- 
sions and  bacterial  action  prevented  by  the  addition  of  0.2  cubic 
centimeter  of  toluol.  The  casein  was  precipitated  from  these  at  inter- 
vals by  adding  20  cubic  centimeters  of  1  per  cent  acetic  acid  and  the 
nitrogen  determined  as  above.  The  data  showing  autohydrolysis 
will  be  submitted  first. 

Autohydrolysis  of  casein  in  solution. 
[Average  of  2  samples.] 


Period  of  incubation. 


N  present. 


N  precipi- 
tated. 


N  hydro- 
lyzed. 


Per  cent  of 

Nhydro- 

lyzed. 


lhour 
3  days 
9  days 


Mg. 


94.3 
94.3 
94.3 


Mg. 


90.4 
89.6 
87.6 


Mg. 


3.9 
4.7 
6.4 


4.13 
4.96 
6.79 


The  above  data  show  that  in  one  hour's  time  4.13  per  cent  of  the 
nitrogen  underwent  autohydrolysis,  and  this  was  increased  upon 
standing  for  9  days  to  6.79  per  cent. 

The  effects  of  bacterial  action  on  the  ammonification  and  hydrol- 
ysis of  casein  are  shown  as  follows : 

Some  results  of  bacterial  action  on  casein. 
[Average  of  2  samples.] 


Period  of  incubation. 


lday. 

3  days 

4  days 

5  days 
7  days 
9  days 


N  added. 


Mg. 


94.3 
94.3 
94.3 
94.3 
94.3 
94.3 


N  found 
asNH3. 


Mg. 

0.0 

.6 

2.5 

9.7 

56.1 

58.7 


N  precipi- 
tated. 


Mg. 


90.4 
91.1 
75.9 
29.6 
13.8 
17.6 


Per  cent  of 
N  ammoni- 
fied. 


0.00 
.64 

2.65 
10.28 
59. 53 
62.25 


Per  cent  of 
N  hydro- 
lyzed. 


4.13 
3.39 
19.51 
68.61 
85.36 
81.32 


Active  ammonification  set  in  after  the  fourth  day  and  reached  a 
practical  maximum  on  the  seventh  day,  when  59.53  per  cent  of  the 
nitrogen  had  been  converted  into  ammonia.  Active  hydrolysis  set 
in  after  the  third  day  and  was  completed  by  the  seventh  day.  By 
this  time  the  solutions  had  become  quite  opalescent,  due  to  the 
abundance  of  cells  of  bacteria  and  fungi,   and  no  precipitate  was 


Jour.  Biol.  Chem.,  11  (1912),  pp.  267-305. 


17 

formed  upon  acidifying  with  acetic  acid.  The  nitrogen  recorded  as 
precipitated  on  the  seventh  and  ninth  days,  therefore,  was  not  in  the 
form  of  casern  but  was  contained  in  the  bacterial  cells  that  were  held 
up  by  the  filter  paper. 

From  the  foregoing  it  is  apparent  that  bacterial  hydrolysis  of 
casein  precedes  ammonification  and  that  the  former  takes  place  con- 
siderably more  rapidly  than  the  latter. 

EFFECTS  QF  BACTERIAL  ACTION  ON   DIFFERENT   GROUPS  OF 
NITROGEN  COMPOUNDS. 

In  the  preceding  experiments  maximum  ammonification  usually 
took  place  in  from  four  to  six  days.  Of  the  substances  tested  casein 
was  the  most  completely  ammonified,  but  as  stated  above,  usually 
not  more  than  60  per  cent  of  the  nitrogen  was  converted  into  ammonia. 

The  following  experiments  were  made  for  the  purpose  of  studying 
the  effects  of  bacterial  action  on  the  different  groups  of  organic  nitro- 
gen compounds.  Two  gram  portions  pf  the  substances  were  mixed 
with  100  grams  of  silica  sand,  soil  infusions  added  and  incubated  for 
definite  periods.  Then  the  sand  mixtures  were  transferred  to  1,000 
cubic  centimeter  Kjeldahl  flasks,  400  cubic  centimeters  of  hydro- 
chloric acid  added,  and  the  whole  boiled  under  reflux  condensers  for 
10  hours.  After  filtering  and  washing  the  residue  with  hot  water,  the 
filtrates  were  diluted  to  1,000  cubic  centimeters  and  aliquots  used  in 
the  determination  of  the  amid,  basic,  and  nonbasic  groups  of  nitrogen 
compounds,  employing  the  same  methods  as  were  used  in  previous 
work  on  the  organic  nitrogen  of  Hawaiian  soils.1 

In  every  case  the  residues  left  after  filtration  were  practically  free 
from  nitrogen,  showing  that  all  the  nitrogen  present  went  into  solu- 
tion, but  a  smaller  amount  was  generally  found  than  occurred  in  the 
original  materials.  This  was  probably  due  to  the  loss  of  ammonia 
by  volatilization  during  the  course  of  bacterial  action,  and  to  the 
decomposition  of  nitrates,  and  therefore,  will  be  considered  as  having 
been  converted  into  ammonia.  It  is  possible,  however,  that  some 
denitrification  also  took  place.  The  length  of  time  that  the  different 
materials  were  exposed  to  bacterial  action  varied,  the  purpose  being 
to  allow  decomposition  to  continue  no  longer  than  was  necessary  to 
insure  vigorous  ammonification.  Ammonia  was  determined  in 
separate  portions  by  direct  distillation  with  magnesia.  The  original 
materials  were  also  subjected  to  acid  hydrolysis  and  the  group  deter- 
minations made  as  above.  All  determinations  were  made  in 
duplicate. 

The  materials  studied  include  casein,  dried  blood,  soy  bean  cake 
meal,  cottonseed  meal,  and  linseed   meal  whose  nitrogen  contents 

i  Hawaii  Sta.  Bui.  33  (1914). 


18 

have  already  been  given.  Coconut  meal,  containing  3.30  per  cent 
nitrogen,  globulin  from  cottonseed  meal,  containing  16.38  per  cent 
nitrogen,  and  zein  from  maize,  containing  14.03  per  cent  nitrogen, 
were  also  used.  Neither  the  globulin  nor  the  zein  was  entirely  pure, 
as  is  shown  by  the  nitrogen  content. 

casein. 

Nitrogen  content  of  casein  and  its  bacterial  decomposition  products. 


Per  cent  of  original 
material. 

Per  cent  of  total  N. 

Per  cent  of 
groups  de- 
composed. 

Per  cent  of 
organic  N 

Before. 

After. 

Before. 

After. 

after  bacte- 
rial action. 

3.93 
1.10 
0.95 
6.31 

31.96 

8.88 
7.67 
50.93 

Amid  N 

1.54 
2.12 

8.87 

12.43 
17.11 
71.59 

28.57 
59.19 

28.86 

13  16 

Basic  N 

11  34 

75  48 

The  casein  used  was  not  pure.  In  addition  to  considerable  ether 
soluble  matter  it  probably  contained  small  amounts  of  nitrogen 
bodies  other  than  casein.  Pure  casein  from  cow's  milk  contains 
10.3  per  cent  of  the  nitrogen  in  the  form  of  amids  and  22.4  per  cent 
as  basic  nitrogen  compounds,  while  the  above  material  yielded  12.43 
per  cent  as  amids  and  only  17.11  per  cent  as  basic  compounds. 

The  bacterial  action  was  allowed  to  continue  for  three  days,  during 
which  time  31.96  per  cent  of  the  total  nitrogen  was  converted  into 
ammonia.  The  amid  nitrogen  was  reduced  from  12.43  per  cent  to 
8.88  per  cent  of  the  total,  basic  nitrogen  from  17.11  per  cent  to  7.67 
per  cent,  and  nonbasic  nitrogen  from  71.59  per  cent  to  50.93  per  cent. 
Expressed  in  percentages  of  decrease  we  find  that  28.57  per  cent  of  the 
amid  nitrogen,  55.19  per  cent  of  the  basic,  and  28.86  percent  of  the 
nonbasic  nitrogen  were  ammonified.  The  organic  nitrogen  remain- 
ing at  the  close  of  the  experiment  was  composed  of  13.16  per  cent 
amid,  11.34  per  cent  basic,  and  75.48  per  cent  nonbasic  nitrogen 
compounds.  Comparing  these  percentages  with  the  composition  of 
the  original  casein  it  will  be  seen  that  the  basic  nitrogen  compounds 
were  decomposed  more  rapidly  than  the  amids  or  nonbasic  nitrogen 
compounds. 

DRIED    BLOOD. 

Nitrogen  content  of  dried  blood  and  its  bacterial  decomposition  products. 


Per  cent  of  original 
material. 

Per  cent  of  total  N. 

Per  cent  of 
groups  de- 
composed. 

Per  cent  of 
organic  N 
after  bacte- 
rial action. 

Before. 

After. 

Before. 

After. 

6.11 

.67 

1.09 

5.65 

45.19 
4.95 
8.06 

41.76 

Amid  N 

1.23 
2.52 
9.77 

9.09 
18.64 
72.26 

45.53 
56.75 
42.17 

9.04 

Basic  N 

14.71 

Nonbasic  N 

76.25 

19 

The  dried  blood  underwent  bacterial  decomposition  for  seven  days 
and  45.19  per  cent  of  the  nitrogen  was  ammonified.  The  amid  nitro- 
gen decreased  from  9.09  per  cent  to  4.95  per  cent  of  the  total  nitrogen, 
the  basic  nitrogen  from  18.64  per  cent  to  8.06  per  cent,  and  the  non- 
basic  nitrogen  from  72.26  per  cent  to  41.76  per  cent.  Calculating  the 
percentages  of  decomposition  in  the  different  groups  we  find  that 
45.53  per  cent  of  the  amid  nitrogen,  56.75  per  cent  of  the  basic 
nitrogen,  and  42.17  per  cent  of  the  nonbasic  nitrogen  were  ammonified. 
These  data  show  that  the  basic  diamino  acids  were  decomposed  more 
rapidly  than  the  other  groups.  The  organic  nitrogen  remaining  after 
the  bacterial  action  was  composed  of  9.04  per  cent  amid,  14.71  per 
cent  basic,  and  76.25  per  cent  nonbasic  nitrogen  compounds,  as  com- 
pared with  9.09  per  cent  amid,  18.64  per  cent  basic,  and  72.26  per 
cent  nonbasic  nitrogen  in  the  original  dried  blood. 


SOY    BEAN    CAKE    MEAL. 


The  bacterial  decomposition  of  this  material  took  place  for  five 
days,  with  the  results  as  given  below: 

Nitrogen  content  of  soy  bean  cake  meal  and  its  bacterial  decomposition  products. 


Per  cent  of  original 
material. 

Per  cent  of  total  N. 

Per  cent  of 
groups  de- 
composed. 

Per  cent  of 
organic  N 
after  bacte- 
rial action. 

Before. 

After. 

Before.   |     After. 

Ammonia  N 

4.10 
.69 
.24 

3.25 

49.52 

AmidN 

1.24 

.76 

6.28 

14.97 
9.18 
75.84 

8.33 

2.89 

39.25 

43.06 
67.10 
48.25 

16.51 

Basic  N 

5.74 

Nonbasic  N 

77.75 

The  above  table  shows  that  49.52  per  cent  of  the  total  nitrogen 
was  ammonified.  The  amid  nitrogen  decreased  from  14.97  per  cent 
to  8.33  per  cent  of  the  total,  the  basic  diamino  acids  from  9.18  per 
cent  to  2.89  per  cent,  and  the  nonbasic  nitrogen  from  75.84  per  cent 
to  39.25  per  cent.  Expressed  in  percentages  of  decomposition  it  is 
found  that  43.06  per  cent  of  the  amid  nitrogen,  67.10  per  cent  of  the 
basic  nitrogen,  and  48.25  per  cent  of  the  nonbasic  nitrogen  were 
ammonified.  The  organic  nitrogen  remaining  was  composed  of 
16.51  per  cent  amid,  5.74  per  cent  basic,  and  77.75  per  cent  nonbasic 
nitrogen  compounds,  as  compared  with  14.97  per  cent  amid,  9.18 
per  cent  basic,  and  75.84  per  cent  nonbasic  nitrogen  in  the  original 
material. 


20 


COTTONSEED   MEAL. 


Cottonseed  meal  was  exposed  to  bacterial  action  for  eight  days, 
with  the  results  shown  in  the  following  table: 

Nitrogen  content  of  cottonseed  meal  and  its  bacterial  decomposition  products. 


Per  cent  of  original 
material. 

Per  cent  of  total  N. 

Per  cent  of 
groups  de- 
composed. 

Per  cent  of 
organic  N 
after  bacte- 
rial action. 

Before. 

After. 

Before. 

After. 

1.45 

0.59 

.32 

2.74 

28.43 
11.57 
6.27 
53.72 

Amid  N 

0.78 

.96 

3.36 

15.29 

18.83 
65.88 

24.37 
66.67 
15.48 

16.15 

Basic  N 

8.77 

Nonbasic  N 

75.07 

The  above  data  show  that  28.43  per  cent  of  the  total  nitrogen  was 
ammonified.  The  amid  nitrogen  decreased  from  15.29  per  cent  to 
11.57  per  cent  of  the  total,  the  basic  nitrogen  from  18.83  per  cent  to 
6.27  per  cent,  and  the  nonbasic  nitrogen  from  65.38  per  cent  to 
53.72  per  cent.  Expressed  in  percentages  of  decomposition  it  was 
found  that  24.37  per  cent  of  the  amid  nitrogen,  66.67  per  cent  of  the 
basic  nitrogen,  and  15.48  per  cent  of  the  nonbasic  nitrogen  were 
ammonified.  Thus  it  is  found  that  the  basic  diamino  acids  were 
ammonified  more  rapidly  than  the  other  groups. 

LINSEED   MEAL. 

Linseed  meal  was  subjected  to  bacterial  action  for  seven  days, 
with  the  following  results: 

Nitrogen  content  of  linseed  meal  and  its  bacterial  decomposition  products. 


Per  cent  of  original 
material. 

Per  cent  of  total  N. 

Per  cent  of 
groups  de- 
composed. 

Per  cent  of 
organic  N 
after  bacte- 
rial action. 

Before. 

After. 

Before. 

After. 

1.99 

.64 
.38 
1.99 

39.80 

12.80 

7.60 

39.80 

Amid  N 

0.83 

.62 

3.55 

16.60 
12.40 
71.00 

22.89 
37.09 
43.66 

21.26 

Basic  N 

12.62 

Nonbasic  N 

66.11 

It  will  be  seen  from  the  above  table  that  39.80  per  cent  of  the 
nitrogen  was  ammonified.  The  percentages  of  decomposition  show 
that  22.89  per  cent  of  the  amid  nitrogen,  37.09  per  cent  of  the  basic 
nitrogen,  and  43.66  per  cent  of  the  nonbasic  nitrogen  were  ammonified. 
The  organic  nitrogen  remaining  was  composed  of  21.26  per  cent 
amid,  12.62  per  cent  basic,  and  66.11  per  cent  nonbasic  compounds, 
as  compared  with  16.60  per  cent,  12.40  per  cent,  and  71  per  cent, 
respectively,  in  the  original  material.     Thus  it  is  shown,  in  contrast 


21 

to  the  materials  reported  above,  that  the  nonbasic  monamino  acids 
of  linseed  meal  were  decomposed  more  rapidly  than  the  nitrogen 
compounds  of  other  groups. 

COCONUT   MEAL. 

Preliminary  ammonification  experiments  with  this  material  indi- 
cated that  the  nitrogen  constituents  would  be  decomposed  more 
slowly  than  in  the  materials  reported  above.  After  incubating 
one  week  practically  no  ammonia  was  found.  Consequently  the 
decomposition  was  allowed  to  take  place  for  12  days,  but  even  then 
only  a  small  amount  of  ammonia  was  formed. 

Nitrogen  content  of  coconut  meal  and  its  bacterial  decomposition  products. 


Per  cent  of  original 
material. 

Per  cent  of  total  N. 

Per  cent  of 
groups  de- 
composed. 

Per  cent  of 
organic  N 
after  bacte- 
rial action. 

Before. 

After. 

Before. 

After. 

0.24 

.37 

.41 

2.28 

7.27 
11.21 
12.  43 
69.09 

Amid  N 

0.38 

.52 

2.40 

11.52 
15.76 
72.72 

2.67 
21.15 
5.00 

12.09 

Basic  N 

13.39 

Nonbasic  N 

74.51 

It  would  seem  that  the  carbohydrates  and  fats  protected  the 
nitrogen  bodies  from  bacterial  decomposition,  since  only  7.27  per 
cent  of  the  total  nitrogen  was  found  as  ammonia,  and  the  absolute 
amounts  of  nitrogen  in  the  different  groups  were  only  slightly 
different  from  those  in  the  original  material.  But  the  magnitude 
of  the  experimental  error  was  relatively  too  great  to  justify  positive 
conclusions. 

The  data  show,  however,  that  a  higher  percentage  of  the  ammonia 
was  derived  from  the  basic  nitrogen  group  than  in  any  of  the  previous 
experiments. 

GLOBULIN    FROM    COTTONSEED    MEAL. 

The  globulin  was  prepared  from  cottonseed  meal  by  extraction 
with  a  10  per  cent  solution  of  sodium  chlorid,  then  precipitated  by 
saturating  the  solution  with  ammonium  sulphate,  redissolved  in 
sodium  chlorid  solution,  and  dialyzed.  The  product  was  washed 
with  alcohol  and  ether,  and  dried  in  vacuum  over  sulphuric  acid, 
but  was  still  impure  as  shown  by  the  nitrogen  content.  Ammonifi- 
cation continued  for  three  days. 


22 

Nitrogen  content  of  globulin  from  cottonseed  meal  and  its  bacterial  decomposition  products. 


Per  cent  of  original 
material. 

Per  cent  of  total  N. 

Per  cent  of 
groups  de- 
composed. 

Per  cent  of 
organic  N 
after  bacte- 
rial action. 

Before. 

After. 

Before. 

After. 

Ammonia  N 

4.55 
1.04 

1.87 
8.92 

27.77 

6.35 

11.41 

54.46 

Amid  N 

1.80 
3.77 
10. 81 ' 

10.99 
23.02 
65.99 

42.22 
50.39 
17.48 

8.81 

Basic  N 

15.80 

Nonbasic  N 

75.40 

The  nitrogen  of  'the  original  material  was  composed  of  10.99 
per  cent  amid,  23.02  per  cent  basic,  and  65.99  per  cent  nonbasic 
compounds.  Pure  globulin  from  cotton  seed,  on  the  other  hand, 
contains  10.3  per  cent  amid,  30.6  per  cent  basic,  and  59.1  per  cent 
nonbasic  nitrogen.  It  is  possible  that  the  low  percentage  of  basic 
nitrogen  and  the  correspondingly  high  percentage  of  nonbasic  nitrogen 
found  above  were  due  to  incomplete  hydrolysis,  as  Osborne  has 
pointed  out  that  some  vegetable  proteins  require  continuous  boiling 
for  24  hours  for  complete  hydrolysis. 

The  above  data  show  that  27.77  per  cent  of  the  nitrogen  was 
converted  into  ammonia,  and  that  42.22  per  cent  of  the  amid,  50.39 
per  cent  of  the  basic,  and  17.48  per  cent  of  the  nonbasic  nitrogen 
compounds  were  decomposed.  The  organic  nitrogen  remaining 
after  bacterial  action  was  composed  of  8.81  per  cent  amid,  15.80 
per  cent  basic,  and  75.40  per  cent  nonbasic  compounds.  Comparing 
the  above  data  with  that  obtained  with  the  use  of  cottonseed  meal, 
it  is  of  special  interest  to  note  that  globulin,  when  separated  from 
the  other  nitrogen  and  nonnitrogenous  constituents  of  cottonseed 
meal,  undergoes  bacterial  decomposition  in  very  much  the  same 
way  as  do  the  nitrogen  compounds  of  cottonseed  meal  as  a  whole. 
In  each  instance  the  basic  diamino  acids  were  decomposed  more 
rapidly  than  the  other  groups.  The  amids,  however,  were  decom- 
posed more  rapidly  in  the  globulin  than  in  cottonseed  meal. 

ZEIN    FROM   MAIZE. 

With  the  exception  of  linseed  meal  the  basic  diamino  acids  of  the 
preceding  materials  were  decomposed  more  rapidly  than  the  amids  or 
monamino  acids.  The  basic  nitrogen  in  these  materials  varied  from 
9.18  per  cent  to  23.02  per  cent  of  the  total  nitrogen.  In  order  to  study 
the  decomposition  of  a  substance  containing  still  less  diamino  nitrogen, 
zein  was  prepared  from  maize  by  alcoholic  extraction.  The  product 
was  not  highly  purified  but  the  analysis  shows  that  practically  all 
the  nitrogen  was  in  the  form  of  zein. 


23 

Nitrogen  content  ofzeinfrom  maize  and  its  bacterial  decomposition  products. 


• 

Per  cent  of  original 
material. 

Per  cent  of  total  N. 

Per  cent  of 
groups  de- 
composed. 

Per  cent  of 
organic  N 
after  bacte- 
rial action. 

Before. 

After. 

Before. 

After. 

Ammonia  N 

0.98 

2.21 

.38 

10.46 

6.98 
15.75 

2.78 
74.55 

AmidN 

2.75 

.43 

10.85 

19.60 
3.06 
77.33 

19.64 
11.63 
3.59 

16.93 

2  91 

Nonbasic  N 

80.15 

Bacterial  action  was  allowed  to  continue  for  three  days,  but,  as 
shown  in  the  table,  only  small  amounts  of  ammonia  were  formed. 
The  zein  as  prepared  was  in  a  tough,  horny  condition,  and  was  conse- 
quently difficult  to  pulverize,  which  probably  accounts  in  part  for  the 
low  yields  of  ammonia.  The  data  show  that  the  basic  diamino  acids 
were  only  decomposed  to  a  slight  extent;  but  in  this  case  the  amids 
were  most  markedly  decomposed.  The  decreases  in  amid  nitrogen 
as  determined,  however,  may  not  have  been  entirely  due  to  ammoni- 
fication,  since  any  amid  compounds  that  were  split  off  by  the  bac- 
teria, but  not  ammonified,  would  have  been  decomposed  and  deter- 
mined as  ammonia  along  with  that  actually  formed  by  the  bacteria. 

As  shown  above,  a  large  portion  of  the  nitrogen  in  the  materials 
used  was  not  ammonified,  or  at  least  did  not  occur  at  any  one  time  as 
ammonia;  neither  was  the  yield  of  ammonia  from  casein  in  the  experi- 
ments with  soil  materially  increased  by  prolonging  the  time  of  decom- 
position beyond  four  days.  But  the  cessation  of  ammonification  was 
not  due  to  the  accumulation  of  poisonous  by-products,  since  the 
second  and  third  grams,  added  after  the  ammonification  of  one  gram 
had  come  to  a  stop,  were  each  ammonified  to  a  slightly  greater  extent 
than  the  first  gram.  It  seems  probable,  therefore,  that  a  part  of  the 
organic  nitrogen  in  the  materials  used  is  more  resistant  to  ammoni- 
fication than  others.  It  should  also  be  remembered  that  putrefac- 
tive decomposition  usually  takes  place  to  some  extent  in  the  ordinary 
ammonification  experiment,  which  probably  results  in  the  formation 
of  the  aromatic  protein  cleavage  products,  tyrosin,  phenylalanin,  and 
tryptophane  at  first;  later  these  are  decomposed  into  indol  and 
skatol,  rather  than  being  immediately  converted  into  ammonia.  It 
seems  also  that  a  portion  of  the  nitrogen  was  assimilated  by  the 
organisms  present,  but  whether  the  assimilation  of  ammonia  or 
organic  forms  took  place  can  not  be  definitely  stated.  The  latter 
seems  the  more  probable.  In  either  case  it  is  reasonably  certain  that 
synthesis  as  well  as  decomposition  plays  a  considerable  part  in  the 
chemistry  of  soil  organic  nitrogen. 

Finally  the  basic  diamino  nitrogen  of  organic  materials  is  ammoni- 
fied, or  otherwise  loses  its  identity  as  such  more  rapidly  than  the 


24 

amids  and  monamino  acids.  This  phase  of  the  chemistry  of  bacterial 
action  is  in  harmony,  therefore,  with  the  indications  given  by  previous 
study  on  the  organic  nitrogen  of  soils. 

SUMMARY. 

(1)  The  ammonification  of  casein  in  silica  sand  was  much  more 
rapid  during  the  first  two  days  than  that  of  dried  blood,  soy  bean 
cake  meal,  cottonseed  meal,  or  linseed  meal,  while  soy  bean  cake 
meal  was  second  in  the  order  of  decomposition.  Later  loss  of  am- 
monia by  evaporation  reduced  the  concentration  of  ammonia,  thus 
making  it  impossible  to  compare  the  rates  of  decomposition. 

(2)  During  the  first  two  days  the  rate  of  ammonification  in  soil 
was  similar  to  that  in  sand,  and  a  much  higher  percentage  of  the  total 
nitrogen  in  casein  was  ammonified  than  of  the  other  materials.  On 
the  ninth  day  50.2  per  cent  of  the  casein  nitrogen,  42.4  per  cent 
in  dried  blood,  40.9  per  cent  in  soy  bean  cake  meal,  27.1  per 
cent  in  cottonseed  meal,  and  26  per  cent  in  linseed  meal  had  been 
ammonified. 

(3)  When  equal  amounts  of  nitrogen  were  added,  casein  still  under- 
went more  rapid  ammonification  during  the  first  two  days  than  the 
other  materials,  and  cottonseed  meal  and  soy  bean  cake  meal  were 
more  completely  ammonified  than  dried  blood  or  linseed  meal.  Later 
the  yield  of  ammonia  from  dried  blood  exceeded  that  from  cottonseed 
meal.  During  the  nine  days  of  the  experiment  56.9  per  cent  of  the 
nitrogen  in  casein,  49.3  per  cent  in  dried  blood,  48.7  per  cent  in  soy 
bean  cake  meal,  32  per  cent  in  cottonseed  meal,  and  34.6  per  cent 
in  linseed  meal  were  ammonified. 

(4)  Under  anaerobic  conditions  all  of  the  materials  were  ammoni- 
fied very  slowly  during  the  first  two  days.  Later  the  casein  was  con- 
verted into  ammonia  approximately  to  the  same  extent  as  under 
aerobic  conditions,  but  the  other  materials  were  decomposed  much 
less  vigorously. 

(5)  With  equal  amounts  of  both  nitrogen  and  nonnitrogenous 
matter  present  the  final  yields  of  ammonia  from  the  different  ma- 
terials, with  the  exception  of  dried  blood,  agreed  closely,  but  the  initial 
decomposition  of  casein  was  still  much  more  active  than  the  other 
substances.  The  yield  of  ammonia  from  casein  on  the  ninth  day  was 
only  31.4  per  cent  as  compared  with  56.9  per  cent  in  the  absence  of 
starch,  and  the  ammonification  of  dried  blood  was  reduced  from  49.3 
per  cent  to  18.9  per  cent.  It  has  been  suggested  that  the  ammoni- 
fying organisms  are  able  to  utilize  carbohydrates  to  some  extent  as 
sources  of  energy.  If  so,  smaller  amounts  of  ammonia  would  conse- 
quently be  split  off  from  proteins  in  the  presence  of  carbohydrates. 
Hence  the  carbon-nitrogen  ratio  would  materially  affect  the  actual 
formation  of  ammonia  in  soils. 


25 

(6)  When  the  amounts  of  casein  were  varied,  other  conditions 
remaining  constant,  the  yields  of  ammonia  in  four  days  increased 
as  the  amounts  of  casein  present  increased;  48.4  per  cent  of  the  total 
nitrogen  in  0.2  gram  was  ammonified,  57  per  cent  in  1  gram,  60.9 
per  cent  in  2  grams,  and  65.9  per  cent  in  3  grams.  It  seems  probable 
that  decreasing  percentages  of  the  total  nitrogen  were  assimilated 
by  the  organisms  present  as  the  amounts  present  increased,  but  there 
are  probably  other  factors  of  a  chemical  and  biological  nature 
involved. 

(7)  The  yield  of  ammonia  from  casein  was  not  materially  increased 
by  extending  the  incubation  period  beyond  four  days,  and  the 
decomposition  of  the  second  and  third  gram,  added  after  one  gram 
had  been  acted  upon  four  and  eight  days,  respectively,  was  slightly 
more  vigorous  than  that  of  the  first  gram.  In  each  instance  approxi- 
mately 60  per  cent  of  the  total  nitrogen  was  found  as  ammonia. 
These  facts,  taken  in  connection  with  the  above,  indicate  that  the 
incomplete  ammonification  was  not  due  to  the  inhibitory  effect  of 
the  decomposition  products,  but  rather  that  a  part  of  the  nitrogen 
of  casein  is  extremely  resistant  to  ammonification.  It  is  also  possi- 
ble that  a  large  part  of  the  remaining  nitrogen  was  assimilated  by 
the  bacteria. 

(8)  Casein  when  mixed  with  silica  sand  or  in  solution  was  com- 
pletely hydrolyzed  by  the  action  of  bacteria  in  seven  days.  In  the 
former  instance,  64.2  per  cent  of  the  nitrogen  was  ammonified  and  in 
the  latter  59.53  per  cent.  In  solution  the  rate  of  hydrolysis  exceeded 
that  of  ammonification,  but  the  latter  was  not  so  active  during  the 
first  five  days  as  when  mixed  with  soil  (see  Series  IV). 

(9)  The  determination  of  the  different  groups  of  nitrogen  com- 
pounds before  and  after  bacterial  action  in  casein,  dried  blood,  soy 
bean  cake  meal,  cottonseed  meal,  linseed  meal,  coconut  meal,  globulin 
from  cottonseed  meal  and  zein  from  maize  shows  that,  with  the 
exception  of  linseed  meal  and  zein,  the  basic  diamino  acid  nitrogen 
was  converted  into  ammonia  more  rapidly  than  the  nitrogen  of  other 
groups.  With  casein,  soy  bean  cake  meal,  and  cottonseed  meal  the 
more  rapid  ammonification  of  the  basic  nitrogen  was  especially 
noticeable.  When  this  fact  and  the  above  are  considered  in  connec- 
tion with  a  comparison  of  the  organic  nitrogen  of  soils  and  vegetable 
proteins,  it  becomes  apparent  that  all  portions  of  the  organic  nitrogen 
in  the  different  materials  used  as  fertilizers  and  green  manures  are  not 
equally  susceptible  to  ammonification.  It  is  evident,  therefore,  that 
chemical  factors  inherent  in  the  nitrogen  compounds  themselves 
predetermine  the  availability  to  some  degree.  Further  investigation, 
including  a  study  of  the  decomposition  of  individual  amino  acids 
and  acid  amids,  is  being  made. 


UNIVERSITY  OF  FLORIDA 

Ijlflllllll 

3  1262  08929  1008 


